Two new hermit crab species of Diogenes (Crustacea: Decapoda: Diogenidae) from Atlanto‐Mediterranean coasts of Iberian Peninsula: Poleward migrants or merely overlooked indigenous species?

Abstract A new hermit crab species of the genus Diogenes with reddish‐orange cheliped, Diogenes erythromanus sp. nov., is described and illustrated based on specimens from the Mediterranean coasts of the Iberian Peninsula, southern Spain. In addition, a second morphotype originating from Mauritanian waters and morphologically very close to D. erythromanus sp. nov. is described as a different species, D. arguinensis sp. nov. The new species are here compared to morphologically similar congeners, especially to those inhabiting the same geographical range. Diogenes erythromanus sp. nov. is distinguishable from other Diogenes primarily by the shape and armature of the left cheliped, with a palm slightly higher than long, with a ridge of spines running along the proximal lower margin that continues with a series of spinose rows forming a central band parallel to the upper margin of the palm. The palm in D. arguinensis sp. nov. is longer than high and shows similar proximal ridge, but without central spinose ridge. The shape of the cheliped is also different in D. arguinensis sp. nov., with long dactylus, which is also flattened and twisted. Sequences from two mitochondrial and one nuclear genes, and comparative analyses with other available sequences for the genus, are also included. Molecular phylogenetic analyses support the morphological delimitation, with D. erythromanus sp. nov. and D. arguinensis sp. nov. forming a separate group, more related to other tropical species, which raises different possible explanations for its presence in the Iberian Peninsula.

The recent revision and comparison of specimens belonging to the genus Diogenes originating from the coasts of the Iberian Peninsula, and nearby areas, revealed the existence of a species complex so far included in the nominal species Diogenes pugilator (Roux, 1829). The study of these material led to the redescription of Diogenes pugilator, the resurrection of D. curvimanus (Clément, 1874), an ancient synonym, and the description of D. armatus Almón et al., 2021 as a new species (Almón et al., 2021), adding two species to the eight already known from the Atlantic. In that work, it was stated that several other morphotypes were identified as different and should be addressed in a separate work.
Some of them were included in the phylogenetic analysis and the results of the phylogeny showed them as valid species and distinguishable from the rest, but without matching any of species known to date.
The present work deals with two of these morphotypes, preliminary labeled as Diogenes sp1 and Diogenes sp2, and has as main objectives: (1) to describe both morphotypes as new species for the genus, (2) to update the information about the genus within European waters, (3) investigate their possible origin and inquire whether their arrival in Europe might be the result of recent migration or otherwise, and (4) to update the identification key for the Atlantic diogenid species.

| MATERIAL S AND ME THODS
Specimens included in this study come from different sources and were obtained during sampling trips conducted during the period 2018-2020. Additional samples were recovered from the crustacean reference collection "Colección de Crustáceos Decápodos y Estomatópodos del Centro Oceanográfico de Cádiz (CCDE-IEOCD)," belonging to the Spanish Institute of Oceanography (IEO-CSIC; Muñoz & García-Isarch, 2021), and from Biological Reference Collections (CBR) at the Institut de Ciències del Mar (ICM-CSIC, Barcelona, Spain; Guerrero et al., 2020), to complete the information about the distribution of these species and to explore the hypothesis of their possible recent arrival to Iberian waters.
All specimens were studied under the stereomicroscope and classified to the lowest taxonomical level possible.
For preventing damage of key structures for morphological identification, a piece of one of the antennae or single ambulatory leg were used as tissue sample for DNA extraction in males and nonovigerous females, while eggs were employed for ovigerous females. Extraction protocol follows that from Estoup et al. (1996) and was carried out at the Instituto de Ciencias Marinas de Andalucía (ICMAN-CSIC). Partial gene sequences were amplified using the following PCR thermal cycles: initial denaturing for 5 min at 95°C; followed by 40 cycles: 30 s at 95°C, 30 s at 45-56°C (depending on primer pairs, see Table 1), 45 s at 72°C; and a final extension of 3 min at 72°C. New specific COI primers (DiogF and DiogR) were designed TA B L E 1 List of primer sequences used in this study for the PCR amplification of partial sequences of 16S, COI and 28S genes, including pair combined, annealing temperature for each primer pair (AT), length of the sequences obtained (bp), and references  Schubart and Huber (2006) COL6b 5′-ACA AAT CAT AAA GAT ATY GG-3′ Schubart and Huber (2006) 28S 28L1 5′-CGG AGG AAA AGA AAC CAA CAG-3′ 28DH2 56 750 Mock and Schubart (2021) 28D2H 5′-TGA CTC GCA CAC ATG TTA GA-3′ 750 Mock and Schubart (2021) for this study due to difficulties in obtaining PCR amplification with the commonly used universal primers. PCR amplicons for partial sequences of the 16S, COI, and 28S genes were sent for purification and sequencing to external laboratories (Stab Vida).
Consensus sequences were generated from the complementary sequences with Bioedit vr. 7.0.5 (Hall, 1999). BLAST searches were performed for each of these sequences in GenBank and for the COI also in BoLD to compare with the available information, confirm or refute the identification, and detect possible issues in these databases.
For the phylogenetic approach, 31 of the available sequences belonging to Diogenes were downloaded from the National Center for Biotechnology Information (NCBI) database ( Table 2) and assembled along with the 153 sequences generated in a previous study (Almón et al., 2021) and the 30 new sequences generated in this study. The final dataset was then aligned by MUltiple Sequence Comparison by Log-Expectation (MUSCLE;Edgar, 2004), implemented in MEGA X version 10.2 .
Blocks of ambiguous data in the non-protein-coding gene alignments were identified and excluded using Gblocks with relaxed settings (Talavera & Castresana, 2007). Gene concatenation of the COI +16S (1187 bp) and the best-fitting nucleotide substitution models for each gene and for the entire alignment were assessed with the tools implemented in MEGA X, using the corrected Akaike information criterion, as recommended by Posada and Buckley (2004). According to the results of this method, the Tamura 3-parameter model of nucleotide substitution using discrete gamma-distributed rates for the variable sites and with invariant sites (T92+G+I) was selected in all cases. Estimates of evolutionary divergence between sequences were obtained using the pairwise distances calculation tool implemented in MEGA X.
Maximum-likelihood (ML) analyses were conducted for the concatenated dataset, as well as for the individual genes (16S, COI, and 28S). Concatenated analyses were partitioned based on gene identity (i.e., 16S and COI). Two species of the family Diogenidae, Dardanus arrosor (Herbst, 1796) and Paguristes eremita (Linnaeus, 1767), were selected as outgroups. ML analyses were performed using MEGA X software under the T92+G+I model. A random starting tree was generated using the Neighbor-Joining method, selecting the partial deletion option (75% site coverage cutoff). A ML tree was generated using the Nearest-Neighbor interchange option. second segment with distolateral outer process stout and acute; smaller but strong spine on distomesial angle; mesial face with 3-4 conspicuous distal spines, with dense short plumose setae associated with the spines; first segment dentate on outer and inner an-

Dardanus arrosor
propodal rasp consisting of 5-6 rows of corneous scales, covering distoventral part including fixed finger. Carpus with distodorsal spine; rest of segments unarmed, with clumps of long plumose setae. var. cristata (Balss, 1921) as described by Rossignol (1962), especially at cheliped level. Despite the undoubted similarities found between the chelipeds, the drawings of the cephalothorax included in his work shows notable differences. Attempts to locate the specimens used by Rossignol in his descriptions have been unsuccessful. In addition, it is difficult to form an opinion on the varieties mentioned or described by Balss (1921) because of the brevity of his diagnoses and the absence of figures. Forest (1955) mentioned that unfortunately the types of the new Balss varieties were destroyed during the war, and that the cristata variety is possibly related to D. denticulatus (and the subcristata variety to a separate species). Therefore, we do not have enough evidence to decide whether or not it could be the same species, which is why the resurrection of the Rossignol variety has been ruled out, describing this form as a new species. On the other hand, according to Rossignol (1957Rossignol ( , 1962, this variety can be easily obtained by diving at shallow depths along the beach of Differentiation of females can be more challenging, with palm of left cheliped in both species being globose with similar shape.
However, the presence of a flat area along the lower margin is persistent in all specimens, making the palm of D. erythromanus sp. nov.
slightly less rounded in shape. Moreover, the outer surface is more granulose, with larger tubercles and spines, while in D. arguinensis sp.
nov. the appearance is smoother, although it does present tubercles, but these are low and rounded.

| Phylogenetic analyses
We analyzed the phylogenetic relationships among the species of

| Biogeographical implications
The presence of D. erythromanus sp. nov. and D. arguinensis sp. nov.
on both sides of the Strait of Gibraltar, as well as the relationships inferred from the phylogenetic analysis, suggest a closest connection with other African congeneric species, rather than with other European ones, opening the possibility to different explanations for their presence in the Iberian Peninsula.

The first hypothesis involves the migration of the species from
Africa to Europe, a process known as tropicalization (Cuesta et al., 2016). It is well known that anthropogenic activities and climate Creep" by Canning-Clode and Carlton (2017). The number of decapod species that have followed this path in the last decades is significant (e.g., Acantharcus posteli (Forest, 1963) (Barnard, 1947)), and is expected that continues to increase in the future (Encarnação et al., 2019;García-Raso, 1985, 1993 There is no clear evidence about which of them may be correct, but according with our samples, it is evident that the southern coast of Spain seems to represent the northern limit of the current distribution for both species.

| Updated key to the presently known Atlantic species of Diogenes Dana, 1851
An update of the recently published "key to the presently known Atlantic species of Diogenes Dana, 1851" (Almón et al., 2021) to include the new species described above, is proposed.

2'
Intercalary rostriform process between ocular acicles smooth 4 3 Ocular peduncles not overreaching base of fifth segment of antennal peduncles. Inner border of antennal acicle concave (see Forest 1955: Fig. 13, Pl. II, fig. 7) D. denticulatus Chevreux & Bouvier, 1892 3' Ocular peduncles long, overreaching base of fifth segment of antennal peduncle. Inner border of antennal acicle straight (see Forest 1961: Fig. 1-4) D. ortholepis Forest, 1961  Palm of male left cheliped higher than long, with well-developed spines on flat area proximoventrally, slightly protruding; outer surface of palm with medial longitudinal row of small spines; dactyl and fixed finger not markedly flattened; lower margin of palm almost straight defined by rows of spines. Pereiopods 2 and 3 clearly overreaching distal margin of left cheliped when fully extended ( Fig. 2A, D present study) D. erythromanus sp. nov.

8'
Palm of male left cheliped longer than high, without protruding flat area proximo-ventrally; outer surface with very short medial row of tubercles; dactyl and fixed finger markedly flattened and twisted; lower margin of palm concave, defined by markedly obtuse spines. Pereiopods 2 and 3 equal in length or slightly overreaching distal margin of left cheliped (Fig.  6A, D, present study) D. arguinensis sp. nov.

9
Palm of male left cheliped clearly longer than high, carpus frequently higher than palm; outer surface of palm finely grained or smooth. Lower margin of carpus of left cheliped long and straight distally, slightly concave proximally. Antennular peduncle shorter than antennal peduncle, ultimate segment widened distally (Forest & Guinot 1956: Fig . 3); (Almón et al., 2021 Figs. 5, 6, 7C,F) D. curvimanus Clément, 1874 (Continues) 9' Palm of male left cheliped not clearly longer than wide, carpus about the same height as palm; outer surface of palm with tubercles or spines, not smooth. Lower margin of carpus of left cheliped convex at distal half, forming a prominent sinus proximally. Antennular peduncle subequal or longer than antennal, not markedly widened distally 10 10 Antennular and antennal peduncles subequal in length. Ocular acicles subtriangular, with 3-5 distal spines (innermost larger), rest of anterolateral outer margin with small tubercles of similar size. Outer surface of left cheliped palm medially inflated, covered with small spinose tubercles; lower inner surface of palm defined by a sinuous crest-like row of large rounded tubercles (Almón et al., 2021 Figs. 1, 2, 7A,D) D. pugilator (Roux, 1829) 10' Antennular peduncles longer than antennal peduncles. Ocular acicles with spines on whole length of anterolateral margin, or few spines on distal half, without tubercles. Outer surface of left cheliped palm not medially inflated, with at least some larger tubercles or spines defining ridges; lower inner surface of palm not defined by a sinuous crest-like row of large rounded tubercles ( The form Diogenes sp. named by Forest in 1956 is not included here, as it has not yet been formally described as a new species. Nevertheless, the form described by Forest can be easily separated from the rest of the Atlantic species by the reduced corneas and ocular peduncles reaching behind distal margin of antennal segment 4. Forest also points out the presence of a conspicuous tooth on disto-outer upper margin of left cheliped palm. a Species with Atlantic records restricted to South African waters only.

| CON CLUS IONS
Studies devoted to the taxonomic revision of the genus Diogenes in the Eastern-Atlantic has been scarce in the last decades, except for the recent studies conducted on South African coasts (Landschoff & Rahayu, 2018). After a golden period of studies on African coasts leaded by J. Forest among others in the mid-19th to 20th centuries (see Barnard, 1950Barnard, , 1955Chevreux & Bouvier, 1892;Forest, 1952Forest, , 1961Kensley, 1981), where most of the current accepted species were described, the account of Diogenes species occurring in this vast area has remained almost inalterable, in part due to the consideration of D. pugilator as a widespread species with high morphological variability (Forest, 1955;McLaughlin et al., 2010).
In a previous work, the examination of the variability of Diogenes species inhabiting a relatively small area as the Iberian Peninsula showed that there were in fact several different species formally assigned to D. pugilator, leading to the description of a new species (D. armatus), the resurrection of an ancient synonymy (D. curvimanus) and the redescription of D. pugilator sensu Roux, 1829(Almón et al., 2021. The redescription of the original species, as well as the designation of neotypes that allow comparison with other specimens, marks a reference point that did not exist until that moment (since the specimens deposited by Roux seem to have been lost), thus providing a stable reference from which to build future studies. The application of an integrative taxonomy approach provides a great opportunity to tackle many ancient conflicts from a new and comprehensive perspective. The species described here also belong to the so-called D. pugilator species complex and adds two new species African coasts, where they are also present. Future studies will contribute to clarify the actual distribution range of the new species, probably revealing along the way, new species still to be discovered.
The revision of the Diogenes species present along European and African coasts is a pending subject that will require cooperative work to overreach the great task that imply the study of this vast and complex area, but at the same time it represents an interesting challenge that will probably yield discoveries that will drastically change the composition and general knowledge about the genus as a whole.

ACK N OWLED G M ENTS
We thank the CBR staff, especially to Pere Abelló and Elena Guerrero for their help in providing specimens for study. This work would not have been possible without the help of Eva García-Isarch and Isabel Muñoz from Centro Oceanográfico de Cádiz (IEO-CSIC) for providing crucially important specimens, and to Carlos Sánchez (ICMAN-CSIC) for his help laboratory work. We also thank the two anonymous reviewers and the editor for their valuable suggestions.
The authors also declare that all materials have been collected under appropriate collection permits and approved ethics guidelines.
These results are part of the PhD thesis of the first author.